Spatiotemporal variability and normalization capability of crAssphage in wastewater-based epidemiology

Wastewater-based epidemiology (WBE) data are commonly normalized to a human fecal indicator to account for the variability in sample composition and analytical efficiency, which can be introduced by environmental or sewer system differences (e.g., infiltration and inflow, wastewater travel time) as well as sample processing. Normalizing pathogen concentrations by a surrogate can theoretically improve cross-site and longitudinal comparability by providing some measures of how some difficult-to-measure factors affect nucleic acid dilution, decay, and recovery. Members of Crassvirales ('crAssphage'), bacteriophages with high human specificity and abundance in the human gut, have been used as a human fecal strength indicator with moderate success in improving data credibility. In this study, we leveraged the wastewater crAssphage quantification data accumulated over four years in New York State. We evaluated the spatiotemporal variability of crAssphage load, and whether crAssphage fulfills dual roles as a normalization factor and quality control for COVID-19 wastewater surveillance. The results suggest that crAssphage load has significant spatiotemporal variability, decreasing its potential as a normalizing factor for WBE. Normalizing SARS-CoV-2 wastewater concentrations to crAssphage had very limited effect on the correlation between SARS-CoV-2 concentration and COVID-19 clinical measures. However, we found that the threshold-exceeding values and non-detection of crAssphage may serve as a quality control step for SARS-CoV-2 surveillance. Our findings refine how the co-quantification of crAssphage can assist WBE.